RESUMEN
A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation. In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Ser/Thr phosphorylation reflected the activation of protein (Ser/Thr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (Ser/Thr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effectors. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (Ser/Thr) kinases to execute their cellular programs. The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance of the Ras-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Proteínas 14-3-3 , Animales , Membrana Celular/metabolismo , Activación Enzimática , Humanos , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Proto-Oncogenes Mas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Insulina/metabolismo , Serina/metabolismo , Transducción de Señal , Treonina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adhesión Celular , Proteínas de Ciclo Celular/metabolismo , Membrana Celular , Clonación Molecular , Citoesqueleto/ultraestructura , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Uniones Intercelulares , Mutagénesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Quinasas p21 ActivadasRESUMEN
The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Animales , Transporte Biológico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Línea Celular , Dimerización , Activación Enzimática , Fibroblastos , Proteína Quinasa 1 Activada por Mitógenos , Modelos Moleculares , Mutación , Fosforilación , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Sitios de Unión , Cristalografía , Dimerización , Activación Enzimática , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Fosforilación , Prolina/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Treonina/metabolismo , Tirosina/metabolismoRESUMEN
Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Transducción de Señal , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , MAP Quinasa Quinasa 2 , Proteína Quinasa 1 Activada por Mitógenos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
To examine the specificity of MEKs for MAP kinase family members, we determined the abilities of several MEK isoforms to phosphorylate mutants of the MAP kinase ERK2 and the related kinase ERK3 which are modified in the phosphorylation loop. The ERK2 mutants included mutations of the two phosphorylation sites, mutations of the acidic residue between these two sites, and mutations that shorten the length of this loop. All mutants were tested for phosphorylation by six mammalian MEKs and compared with several wild type MAP kinases. MEK1 and MEK2 phosphorylate a majority of the ERK2 mutants. MEK2 but not MEK1 will phosphorylate ERK3. Alteration of the residue between the two phosphorylation sites neither dramatically affected the activity of MEK1 and MEK2 toward ERK2 nor conferred recognition by other MEKs. Likewise, reduction of the length of the phosphorylation loop only partially reduces recognition by MEK1 and MEK2 but does not promote recognition by other MEKs. Thus other yet to be identified factors must contribute to the specificity of MEK recognition of MAP kinases.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Activación Enzimática , Datos de Secuencia Molecular , Mutagénesis , Fosforilación , Proteínas Quinasas/genética , Especificidad por SustratoRESUMEN
In the present study we show that purified bovine brain dynamin can be phosphorylated by MAP kinase, ERK2, with a stoichiometry of 1 mol phosphate/mol dynamin. The phosphorylated serine residue is located within the C-terminal 10 kDa of dynamin. Dynamin I phosphorylated by ERK2 can be specifically dephosphorylated by calcineurin but not by protein phosphatase 2A (PP2A). Phosphorylation of dynamin by ERK2 weakens the binding of dynamin to microtubules and inhibits dynamin's microtubule-activated GTPase activity. Stimulation of GTPase activity by either Grb2 or phospholipids was not affected by ERK2 phosphorylation, suggesting that the binding sites for Grb2 and phospholipids do not overlap with that for microtubules.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , GTP Fosfohidrolasas/metabolismo , Microtúbulos/metabolismo , Sitios de Unión , Calcineurina , Proteínas de Unión a Calmodulina/metabolismo , Dinamina I , Dinaminas , Proteína Quinasa 1 Activada por Mitógenos , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2RESUMEN
Rab3 proteins are small GTP-binding proteins of the Ras superfamily. Four highly homologous Rab3 proteins termed Rab3A, Rab3B, Rab3C, and Rab3D have been described. Rab3A has previously been shown to be a constituent of synaptic vesicles in neurons that undergoes membrane dissociation-association cycles during synaptic vesicle recycling. Here we report that Rab3C copurifies with Rab3A during the isolation of synaptic vesicles. Organelles immunoisolated with monoclonal antibodies directed against Rab3A led to a coenrichment of Rab3A and Rab3C, demonstrating that both Rab3 proteins are colocalized on the same organelle. In isolated nerve terminals, stimulation of neurotransmitter release resulted in a dissociation of Rab3C from synaptic vesicle and/or recycling membranes. This dissociation parallels that of Rab3A observed under the same conditions. In contrast, no change was observed in the membrane-association of Rab5, a Rab protein localized on early endosomes. We conclude that in the nervous system Rab3C is localized on synaptic vesicles and, like Rab3A, cycles on and off the synaptic vesicle membrane in parallel with exocytotic release of neurotransmitter.
Asunto(s)
Corteza Cerebral/metabolismo , Exocitosis , Proteínas de Unión al GTP/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/aislamiento & purificación , Immunoblotting , Datos de Secuencia Molecular , Orgánulos/metabolismo , Péptidos/síntesis química , Péptidos/inmunología , Reacción en Cadena de la Polimerasa , Conejos/inmunología , Ratas , Transfección , Proteínas de Unión al GTP rab3RESUMEN
alpha-Latrotoxin is a vertebrate neurotoxin from black widow spider venom that causes massive neurotransmitter release. In order to gain insight into the mechanism of action of alpha-latrotoxin, we have studied alpha-latrotoxin-binding proteins from bovine and rat brain. Proteins purified by affinity chromatography on immobilized alpha-latrotoxin were investigated. Two sets of proteins were isolated: 1) three polypeptides of M(r) 79,000, 65,000, and 43,000 that were eluted from immobilized alpha-latrotoxin by increasing KCl concentrations in the presence of Ca2+, and 2) a family of related proteins ranging in molecular weight from 160,000 to 220,000 and a low molecular weight component of M(r) 29,000 that were eluted from immobilized alpha-latrotoxin only after removal of Ca2+. Amino acid sequences of these proteins demonstrated that all of these proteins represent novel proteins except for the M(r) 65,000 polypeptide, which is identical with synaptotagmin (Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Südhof, T. C. (1991) Nature 353, 65-68). Surprisingly, the M(r) 79,000 and 43,000 proteins were also found in tissues insensitive to alpha-latrotoxin action. Since these proteins do not bind 125I-alpha-latrotoxin with high affinity, their purification probably is not physiologically significant. On the other hand, the fractions containing the M(r) 160,000-220,000 and 29,000 polypeptides bound alpha-latrotoxin with high affinity. Sucrose gradient centrifugations and anion exchange chromatography suggested that most of the M(r) 160,000-220,000 proteins were complexed with the M(r) 29,000 protein. alpha-Latrotoxin binding correlated with the presence of the M(r) 160,000-220,000 proteins and M(r) 29,000 polypeptide, and alpha-latrotoxin formed stable complexes with the M(r) 160,000-220,000 proteins. Accordingly, the alpha-latrotoxin receptor consists of a high molecular weight protein (M(r) 160,000-220,000) that is complexed with one or several copies of an M(r) 29,000 polypeptide. In addition, the receptor is found in a less tight association with synaptotagmin but not with other polypeptides.